We next examined the antiproliferative effects of 20(S,R)-Rg3 or Rk1/Rg5 mixtures, which were collected using preparative HPLC. As shown in Fig. 5A, 5B, 20(S,R)-Rg3 reduced cancer cell viability stronger than Rk1/Rg5 mixture, and each IC50 value were 23.6 μg/mL and 42.9 μg/mL, respectively. Interestingly, the efficacy of 20(S,R)-Rg3 was similar with that of the methanol eluate, as well as of heat-processed Rb1 (Figs.  3D and 4A). To further confirm the main

active component, anticancer effects of 20(S)-Rg3 and 20(R)-Rg3 were individually examined. Subsequently, ginsenoside 20(S)-Rg3 was obviously identified as the main active component of HAG, while there was no effect in ginsenoside 20(R)-Rg3 ( Fig. 5C and D). Thus, anticancer efficacy of HAG was thought LDN-193189 concentration to be mainly related to ginsenoside 20(S)-Rg3, which was transformed from ginsenoside

Rb1 during heat processing. Apoptosis is recognized as an essential mechanism of physiological cell death, and caspases play pivotal roles in cell apoptosis. In line with this Regorafenib order notion, we investigated whether ginsenoside 20(S)-Rg3-induced cell death is involved in apoptosis. A Western blot analysis was first used to evaluate the expression of proteins involved in the apoptotic response to determine if apoptosis occurs via the intrinsic or extrinsic pathway ( Fig. 6A–C). Exposure to ginsenoside 20(S)-Rg3 for 24 h induced the cleavage of PARP, as well as that of caspase-3, caspase-8, and caspase-9, in a dose-dependent manner. In addition, ginsenoside 20(S)-Rg3 significantly triggered the downregulation of Bcl-2 and upregulation of Bax in a dose-dependent manner. Next, we examined the effect of the pan-caspase inhibitor Z-VAD-fmk on cell proliferation to confirm the role played by caspases in ginsenoside 20(S)-Rg3-induced apoptosis. As shown in Fig. 6D, pretreatment with 60 μM Z-VAD-fmk abrogated apoptotic

cell death induced by the ginsenoside 20(S)-Rg3, although the recovery was weak at the high concentration of 50 μg/mL. These findings demonstrate that ginsenoside 20(S)-Rg3 check details induces the activation of caspase-3, caspase-8, and caspase-9, which contributes to apoptotic cell death. Ginsenosides 20(S)-Rg3 and 20(R)-Rg3 are epimers of each other depending on the position of the hydroxyl group (OH) on carbon-20 ( Fig. 1), and this epimerization is known to be produced by the selective attack of the OH group after the elimination of glycosyl residue at carbon-20 during the steaming process [20]. In the present study, 20(S)-Rg3 showed stronger anticancer activity than 20(R)-Rg3. Therefore, stereospecificity exists in the anticancer activity of ginsenoside Rg3 epimers. In addition, stereospecificity in the medicinal efficacy of these ginsenosides has been reported by several researchers.